Normal formation and function of spinal cord circuits requires differentiation of sensory, motor and interneuron subtypes. Our work addresses the role that voltage-gated potassium (Kv) channels play in neuronal subtype differentiation in the embryonic vertebrate spinal cord. We propose that a select subset of Kv channels regulate neuronal differentiation by determining electrical membrane properties that control spontaneous elevations of intracellular calcium, known as calcium (Ca) spikes. Long-duration action potentials occur spontaneously and trigger Ca spikes during a restricted ~6 hr period prior to synapse formation (Stages [St] 20-28). The frequency of Ca spikes determines downstream effects on differentiation programs. We have found that a subset of Kv channels show patterned expression in the embryonic spinal cord and regulate Ca spike properties differently in the dorsal versus ventral embryonic spinal cord. We test the roles of dorsally-expressed Kv channel (Kv1.1) in differentiation of spinal interneurons (Aim 1) and ventrally-expressed Kv channel (Kv2.2) in differentiation of motor neurons (Aim 2). For Aim 3, we focus on subtypes within the motor neuron population and test whether differences in Kv currents account for subtype-specific Ca spike properties and encoded developmental signals Our studies take advantage of the experimental strengths of the Xenopus and zebrafish embryo models and our experience using (1) antisense (AS), morpholino (MO) and dominant negative (DN) overexpression methods in Xenopus and zebrafish embryos, (2) zebrafish genetic mutants and transgenic lines, (3) electrophysiological recording and Ca imaging from neurons in vivo, and (4) morphological methods to analyze spinal cord development. The results of the proposed experiments will provide new insights into molecular mechanisms that allow early spinal cord neuron activity to direct generation of the diverse neuronal identities required for formation of functional circuits.